Successful healing of critical-sized bone defects requires the implantation of a bioactive material that is capable of stimulating vascular infiltration, tissue integration, and normal bone remodeling. To date, engineered tissues consisting of progenitor cells cultured within porous scaffolds have not been as effective as pharmacologic agents for the repair of bone defects, and we postulate that this stems from an insufficient deposition of bioactive factors during in vitro culture. We propose that in vitro perfusion culture of osteoprogenitor cells remains a promising means for achieving clinically effective materials, but that culture strategies conducive to the deposition of osteogenic and angiogenic factors must be identified. Recently, we have found that expression of the osteogenic factor bone morphogenetic protein (BMP)-2 and the angiogenic factor vascular endothelial growth factor (VEGF)-A are induced by perfusion, and that BMP-2 expression in particular is sensitive to pulsatile perfusion. This indicates that mechanotransductive signaling regulates induction of this important bioactive factor, and points to a need to understand the underlying signaling mechanisms, which then may be harnessed to produce bioactive materials. Therefore, the goals of this project are 1) to probe the mechanisms by which pulsatile flow induces expression of BMPs and VEGF-A, and 2) to implement a non-destructive imaging modality to monitor expression of BMP-2 in perfused porous scaffolds. We emphasize that this project will focus on the induction of BMP-2, 4, and 7 and VEGF-A by pulsatile flow. The specific aims of this two-year project are: 1) Determine the effect of pulsatile flow regimens on the induction of BMPs and VEGF, and the activation of discrete signaling pathways. 2) Determine the role of molecular signaling through p38 on the induction of BMPs. 3) Implement bioluminescence computed tomography to image BMP-2 expression in perfused scaffolds. This multi-disciplinary research project integrates tissue engineering, molecular cell biology, and imaging both to advance our fundamental understanding of mechanotransductive signaling pathways, and to develop an enabling technology for tissue engineering. These goals are applied to bone tissue engineering, but have broad applicability to all areas of tissue engineering. The innovative components of this project include planar and 3D perfusion devices to activate mechanotransductive signaling, siRNA technology to probe signaling pathways, and bioluminescence computed tomography to monitor gene induction. PUBLIC HEALTH RELEVANCE Autologous bone graft transplanted from one site in the patient to another is the gold standard material for repair of critical-sized defects, but limited tissue availability and concerns of donor-site complications drive a growing demand for bone substitutes for reconstructive procedures. Sales of bone substitutes were estimated to total $900 million worldwide in 2005, with an annual grows rate of 10% [1]. Recently, orthobiologic materials have emerged as a promising alternative to conventional synthetics, and have a projected growth rate of 74% annually. These materials consist of bioactive proteins that stimulate integration, vascular infiltration, and tissue remodeling embedded within conventional biomaterials (e.g., Infuse Bone Graft, OP-1 Putty [2, 3]). We have extensive evidence that a tissue engineering approach involving culturing of osteoprogenitor cells in porous scaffolds under dynamic perfusion can be used to create a bioactive material (containing osteogenic and angiogenic factors) that may be a tractable alternative to current orthobiologics. The goals of this project are to probe the underlying molecular mechanisms by which perfusion regulates the synthesis of this bioactive matrix, and to develop a non-destructive molecular imaging modality to monitor this synthesis. 1. Artimplant. http://www.artimplant.se/investors/annual_reports. 2005. 2. http://www.fda.gov/cdrh/MDA/DOCS/h020008.html. OP-1 Putty - H020008. 2004. 3. http://www.fda.gov/cdrh/mda/docs/p000058.html. InFUSE" Bone Graft/LT-CAGE" Lumbar Tapered Fusion Device - P000058. 2002.